Microorganism producing frutosyl transferase and method for producing fructooligosaccharides and neofructooligosaccharides using the same

ABSTRACT

The present invention relates to a novel microorganism and a method for producing fructooligosaccharides and neofructooligosaccharides. More particularly, the present invention relates to  Penicillium citrinum  KCTC 10225BP of soil origin which produces fructosyl transferase and hydrolyzes sucrose into fructooligosaccharides of the following formula I: 
                 
 
in which n is integer of 1 to 5, G represents glucose and F represents fructose, and neofructooligosaccharides of the following formula II: 
                 
 
in which n is integer of 1 to 5, G and F are defined as above, at the same time using the fructosyl transferase, and to a method for producing simultaneously fructooligosaccharides and neofructooligosaccharides using said microorganism.

This application claims priority to Korean Patent Application Serial No.2001-0026111, filed May 14, 2001, the contents of which are incorperatedherein, in their entirety, by reference.

TECHNICAL FIELD

The present invention relates to a novel microorganism capable ofsimultaneously producing fructooligosaccharides andneofructooligosaccharides and to a method for producingfructooligosaccharides and neofructooligosaccharides using saidmicroorganism. More particularly, the present invention relates toPenicillium citrinum KCTC 10225BP of soil origin which producesfructosyl transferase and hydrolyzes sucrose into fructooligosaccharidesof the following formula I:

in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose; and neofructooligosaccharides of the followingformula II:

in which n is an integer of 1 to 5, G and F are defined as the above, atthe same time using the fructosyl transferase, and a method forproducing fructooligosaccharides and neofructooligosaccharides usingsaid microorganism.

BACKGROUND ART

Fructooligosaccharides are a mixture of oligosaccharides including1-Kestose (GF2), Nystose (GF3) and Fructosyl nystose (GF4), in which 1to 3 molecule(s) of fructose are bound to sucrose by ®-(2,1) linkage,respectively and are widely contained in plants such as asparagus,onion, potato, honey, etc. They are currently spotlighted as foodmaterials, together with oligosaccharides, due to their outstandingfunctions, including for example, low caloric value, promotion ofproliferation of lactic acid bacteria and bifidobacteria, improvement ofmicroflora in the intestines and inhibition of growth of pathogenicbacteria, improvement of bowel movement, and strengthening of immunity.Thus, examples of applications of oligosaccharides can be found invarious industrial fields, including food, drink, confectionery, healthfood, and the like.

Particularly, it has been reported that the fructooligosaccharidesshowed excellent calcium-absorption effect when used in combination withdifructose anhydride III (DFA III) (See, Japanese Patent Publication No.11-43438). U.S. Pat. No. 5,827,526 disclosed that the duration andrecurrence of diarrhea in a human can be reduced when 0.5 grams to 5grams of fructooligosaccharides per day is administered to the patient.

Korean Patent Application Laid-Open No. 2000-57520 disclosed that amixture of fructooligosaccharides and galactooligosaccharides withvarious edible ingredients may improve the flow in the bowel andeffectively express prebiotic effects, as compared to otheroligosaccharides. Also, currently, fructooligosaccharides are subjectsof research and studies to develop diets for diabetic patients, sincethey are excellent in improvement of bowel movement by inducingproliferation of lactobacillus bifidus, one of the bacteria comprisingnormal microflora of human bowels, do not affect blood sugar levels uponingestion, and are not decomposed by any digestive enzyme. Further, theyare shown to reduce cholesterol levels in the blood and liver.Therefore, such effects of fructooligosaccharides now are not receivingcareful study.

Conventionally, fructooligosaccharides have been produced by methodsusing microorganisms which can prepare fructosyl transferase. There areknown for example, a method using the Aureobasidium pullulans strain, amethod using Aspergillus niger and a method using strains of Penicilliumand Fusarium sps. However, fructosyl transferase prepared by thesemethods has a disadvantage of a low sucrose hydrolysis titer.

Japanese Unexamined Patent Application No. 10-165192 disclosed a methodfor preparing ®-fructofuranosidase using Penicillium citrinum FERMP-15944 fungus, by which a mixture of conventionalfructooligosaccharides with neofructooligosaccharides could be producedfrom sucrose. It is described that the neofructooligosaccharides is amixture consisting of neokestose (6G-®-fructofuranosyl-sucrose),neonystose (6G-®-fructofuranosyl-kestose) and neofructosyl nystose(6G-®-fructofuranosyl-nystose), which have a structure in which 1 to 3molecule(s) of fructose is (are) bound to sucrose by ®- (2,6) linkage,respectively, different from conventional fructooligosaccharides. It isalso described that neofructooligosaccharides have moisturizing effects,excellent sweetness, low calories and anti-cavity effects, functions toinduce proliferation of bacteria in the bowels and to promote topicalimmune responses in the intestinal tracts, and thus can be applicable invarious fields such as sweeteners, functional foods, feed stuffs,medicines, and promoters of pesticides.

D. Grizard et al. disclosed a method for producing a mixture offructooligosaccharides and neofructooligosaccharides using CytolasePCL5, a commercially available enzyme derived from Aspergillus awamori(D. Grizard, C. Barthomuf, Food Biotechnology, 13 (1), 93-105, 1999).

U.S. Pat. No. 5,334,516 disclosed a method for producingneofructooligosaccharides (expressed as “branchedfructooligosaccharides”) with conventional fructooligosaccharides usingan enzyme derived from Aspergillus sydowi.

Thus, the present inventors have intensively investigated and studiedthe prior arts and have conducted research in various ways to producefructooligosaccharides in a high yield. As a result, we have finallyidentified a novel microorganism which is capable of producing fructosyltransferase having a high sucrose hydrolysis titer and confirmed thatconventional fructooligosaccharides and neofructooligosaccharides couldbe produced in a high yield via a reaction of the microorganism with ahighly concentrated sucrose solution. Based on these discoveries, thepresent invention has been developed.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a novelmicroorganism which can fructosyl transferase of a high titer.

It is another object of the present invention to provide a method forproducing fructooligosaccharides and neofructooligosaccharides in highyield by contacting a highly concentrated sucrose solution with themicroorganism.

To accomplish the above objects, the present invention providesPenicillium citrinum KCTC 10225BP of soil origin which producesfructosyl transferase, hydrolyzes sucrose into oligosaccharides of thefollowing formula I:

in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose; and neofructooligosaccharides of the followingformula II:

in which n is an integer of 1 to 5, G and F are defined as the above, atthe same time using the fructosyl transferase, and a method forproducing fructooligosaccharides and neofructooligosaccharides usingsaid microorganism.

In another aspect, the present invention provides a method for producingfructooligosaccharides and neofructooligosaccharides, the methodcomprising the steps of:

-   -   seed culturing the Penicillium citrinum KCTC 10225BP        microorganism according to present invention in a first seeding        medium at 26° C. to 28° C. for 2 days while agitating at a speed        of 100 to 200 rpm to activate the microorganism;    -   mass-producing the microorganism in a fermentation medium at        26° C. to 28° C. for 72 hours while agitating at a speed of 200        to 500 rpm and injecting air at a rate of 0.5 to 1 v/vm; and    -   collecting the produced microorganisms by centrifugation,        washing them twice with 0.85% physiological saline, followed by        culturing in a sucrose solution having a Brix concentration of        sucrose of 60 to 77, as measured by initially inserting a        sensor, at a temperature in a range of 35° C. to 50° C. and pH        of 5 to 7 for 20 to 50 hours, while agitating at a speed of 100        to 300 rpm.

In yet another aspect, the present invention provides a method forproducing fructooligosaccharides and neofructooligosaccharides, themethod comprising the steps of:

-   -   seed culturing the Penicillium citrinum KCTC 10225BP        microorganism according to present invention;    -   mass-producing the seed cultured microorganism;    -   mixing the mass-produced microorganisms with a carrier to form        beads, which are then packed in a column; and    -   passing a sucrose solution through the column packed with the        beads.

In yet another aspect, the present invention provides fructosyltransferase derived from the Penicillium citrinum KCTC 10225BPmicroorganism according to present invention and showing a high sucrosehydrolysis titer.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objects, and other features and advantages of the presentinvention will become more apparent after a reading of the followingdetailed description when taken in conjunction with the drawings, inwhich:

FIG. 1 is a graph showing hydrolysis levels of sucrose while varyingbiomass of Penicillium citrinum KCTC 10225BP according to the presentinvention, wherein the slope (unit/1 g of sucrose) is a sucrosehydrolysis titer of fructosyl transferase;

FIG. 2 a is a graph showing the change in biomass of microorganismsaccording to the fermentation time of the main cultivation using afermentor, while varying the agitation speed from 300 rpm to 500 rpm;

FIG. 2 b is a graph showing the change in the amount of intracellularand extracellular enzymes according to the fermentation time of the maincultivation using a fermentor, while varying the agitation speed from300 rpm to 500 rpm;

FIG. 2 c is a graph showing the change in biomass of the microorganismsand the amount of intracellular enzymes according to the fermentationtime of the main cultivation using a fermentor at the agitation speed of600 rpm;

FIG. 3 a is a graph showing the change in the amount of producedfructooligosaccharides and neofructooligosaccharides according to theconcentration of sucrose in a batch type fermentation after inoculationof Penicillium citrinum KCTC 10225BP according to the present inventioninto 3 of a sucrose solution in a 5 fermentor;

FIG. 3 b is a graph showing the change in the amount of producedfructooligosaccharides and neofructooligosaccharides according to thehydrogen ion concentration (pH) in a batch type production after mixingof biomass of Penicillium citrinum KCTC 10225BP according to the presentinvention into 3 of a sucrose solution in a 5 reactor;

FIG. 3 c is a graph showing the change in the amount of producedfructooligosaccharides and neofructooligosaccharides according totemperature in a batch type production after mixing of biomass ofPenicillium citrinum KCTC 10225BP according to the present inventioninto 3 of a sucrose solution in a 5 reactor;

FIG. 3 d is a graph showing the change in the amount of producedfructooligosaccharides and neofructooligosaccharides according to theagitation speed in a batch type production after mixing of biomass ofPenicillium citrinum KCTC 10225BP according to the present inventioninto 3 of a sucrose solution in a 5 reactor; and

FIG. 4 is a graph showing the change in the amount of producedfructooligosaccharides and neofructooligosaccharides when Penicilliumcitrinum KCTC 10225BP according to the present invention which has beenfixed in alginate is continuously reacted with highly concentratedsucrose solution.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors collected soil distributed around a sugar factorylocated at Inchon, Korea and separated microorganisms from the soil. Oneof the microorganisms was found to show ability to produce fructosyltransferase of a high titer. The novel microorganism was identified asPenicillium citrinum which is a fungus belonging to Penicillium genus.The microorganism was deposited with Korea Research Institute ofBioscience and Biotechnology (KRIBB), located at Oun-dong, Yusong-gu,Taejon, Korea, on Feb. 27, 2001, as Deposition Access No. KCTC-10225BP,so that the microorganism can be available to a third party.

The present inventors examined scientific properties and morphologies ofthe Penicillium citrinum KCTC 10225BP microorganism according to thepresent invention and the results are shown in Table 2. The sucrosehydrolysis titer of fructosyl transferase prepared from themicroorganism was measured. It was found to be 1.5 units/1 g of sucroseon average. Such a result demonstrates that fructosyl transferasederived from Penicillium citrinum KCTC 10225BP according to the presentinvention has a sucrose hydrolysis titer superior to any other knownenzymes, considering the experimental results of U.S. Pat. No.5,334,516, in which when hydrolyzing sucrose using an enzyme derivedfrom Aspergillus, at least 5 units of the enzyme was needed to degrade 1g of sucrose, and D. Grizard et al., in which 7 units of the enzymederived from Aspergillus awamori was needed to degrade 1 g of sucrose.

By using the Penicillium citrinum KCTC 10225BP microorganism accordingto the present invention, it is possible to produce simultaneouslyfructooligosaccharides and neofructooligosaccharides in a high yield.Methods which can be used for the production of fructooligosaccharidesand neofructooligosaccharides include batch type methods, immobilizedcontinuous methods, etc. such as those well-known in the art.

The batch type methods are the most commonly used method, in whichfructosyl transferase are reacted with a sucrose solution (substrate) bymixing microorganisms containing the enzyme with the sucrose solution toobtain a product. In an aspect, the present invention provides a batchtype method for producing fructooligosaccharides andneofructooligosaccharides in a high yield using Penicillium citrinumKCTC 10225BP according to the present invention.

Meanwhile, the immobilized continuous methods, in which microorganismsor enzymes are immobilized in a carrier and a substrate is contactedwith the carrier for reaction with the microorganisms or enzymes, havebeen also widely used up to now. An advantage of these methods is thatmicroorganisms or enzymes can be reused and the reaction can beperformed continuously. However, these methods also have disadvantagesthat for immobilization of enzymes, the enzymes contained inmicroorganisms should be extracted and separated and the enzymesextracted from the microorganisms are generally unstable. Therefore,currently, a method using immobilized microbial cells is used, in whichmicroorganisms are directly immobilized in a carrier. The microbialcells-immobilized method does not require separation of the enzymes fromthe microbial cells and thus, can prevent the reduction of enzymeactivity during the extraction of the enzymes. Also, it has an advantagethat the complicated processes for extraction and separation of enzymescan be performed in a single step. Thus, in another aspect, the presentinvention provides a method for continuously producingfructooligosaccharides and neofructooligosaccharides by immobilizingPenicillium citrinum KCTC 10225BP according to the present invention ina carrier. The immobilized continuous method according to the presentinvention is explained in detail as follows.

First, Penicillium citrinum KCTC 10225BP according to the presentinvention is seed cultured. The seed cultured microorganism ismass-produced in a fermentation medium. The seed culturing is preferablyperformed at 26° C. to 28° C. for 2 days while agitating at a speed of100 to 200 rpm. The mass production is preferably performed at 26° C. to28° C. for 42 to 72 hours while agitating at a speed of 200 to 600 rpmand injecting air at a rate of 0.5 to 1 v/vm. The mass-producedmicroorganisms are then well mixed with a carrier. The mixture is formedinto beads, which are then packed in a column. The carrier is preferablyselected from a group consisting of alginate gel, photo cross-linkedresin, acrylamide gel, |-carrageenan, chitosan and gelatin, thoughcarriers commonly used in the art can be used without limitation. Theconcentration of the carrier is preferably from about 1% to about 2%(w/v). Next, a sucrose solution is passed through the column packed withthe beads. Preferably, the sucrose solution has a concentration of Brix60 to 70 and is flowed at a rate of 100 to 300 mL/hr and a temperatureof 35° C. to 55° C.

Table 1 shows results of two experiments, in whichfructooligosaccharides are produced by a batch type method andimmobilized continuous method using Penicillium citrinum KCTC 10225BPaccording to the present invention.

TABLE 1 Batch Immobilized continuous type method method Biomass ofMicroorganism 400 g 50 g (upon producing 100 L of fructooligosaccharides). Production Efficiency 0.67 L/day 4.02 L/day (uponproducing 100 L of (100 L/150 days) (100 L/24.9 days) fructooligosaccharides).

As seen from the Table 1, when using the batch type method, 400 g ofmicroorganisms was needed to produce 100 L of fructooligosaccharides.The reaction time required to produce 1 L of fructooligosaccharides wasabout 24 hours. However, after completion of the reaction, another 12hours were required until the next reaction was begun in order toseparate the produced fructooligosaccharides from the microorganisms, towash the reaction vessel and to charge the reaction vessel with a freshsucrose solution and microorganisms. On the contrary, in case of theimmobilized continuous method, 50 g of microorganisms was needed toproduce 100 L of fructooligosaccharides. The reaction time required toproduce 100 L of fructooligosaccharides was about 25 days withproduction efficiency of 4.02 L/day. Accordingly, it was confirmed thatwhen applying the immobilized continuous method, a smaller amount ofmicroorganisms is needed and the production efficiency is 6 timeshigher, as compared to the batch type method.

Now, the present invention will be described in detail using anembodiment shown in the following examples. However, the examples arefor illustration of the present invention and do not limit the scope ofthe present invention thereto.

EXAMPLE 1 Identification of Microorganism

Microorganisms were obtained from soil collected around a sugar factorylocated at Inchon, Korea. One of the microorganisms was found to showability to produce fructosyl transferase of a high titer. The novelmicroorganism was identified as Penicillium citrinum which is a fungusbelonging to Penicillium genus. The microorganism was deposited with theKorean Collection for Type Cultures (KCTC), located at Korea ResearchInstitute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong,Yusong-ku, Taejon, 305-333, Republic of Korea, on Feb. 27, 2001, asDeposition Access No. KCTC-10225BP, so that the microorganism can beavailable to a third party.

The microorganism Penicillium citrinum KCTC-10225BP according to thepresent invention were grown in the Czapek-Yeast Algae (CYA) medium andMalt Extract Algae (MEA), commonly used in the art for cultivation offungi. The identified Penicillium citrinum KCTC 10225BP microorganismshowed substantially similar properties and forms when being grown inthe two media. The scientific properties and morphologies of themicroorganism are shown in Table 2 below.

TABLE 2 MEA medium CYA medium Growth characteristics in CYA and MEAmedia Growth rate 2.6 3.6 cm (25° C. for 7 days) communication Growthrate 2.2 cm 2.4 cm (37° C. for 7 days) Growth rate — — 4° C. for 7 days)Surface of Colony Velvet-like, Velvet-like, circular with deep beingflat grooves extending outwardly Mycelium White helical White helicalhypha without hypha without conidium, forming a colony of conidum,Grey-blue-green color forming a colony of Grey-blue- green colorExudates — — Pigment — Forming a pale yellow pigment Bottom surface PaleYellow Dark Yellow of colony Morphological characteristics ConidiophoresGrowing from aerial hypha Sometimes having branches of 20 to 50 μm andgrowing to 100 to 200 μm Smooth surface Foreign spore: furcatum-type,terminating in verticils about 2 to 4 (sometimes monoverticillate)Metulae: branched type and having 5 to 8 phialides, size: 8.6-13.8 ×2.2-3.1 μm Phialide: ampulliform, size: 4.3-8.3 × 1.8-2.9 Conidia Size:2.4-3.2 × 2.0-3.1 μm Spherical Growing in a column shape unwound at theends of conidia

EXAMPLE 2 Seed Cultivation and Measurement of Enzyme Titer

As a medium for use in the seed cultivation of the Penicillium citrinumKCTC 10225BP microorganism, a modification of the medium compositionused in culturing the fructooligosaccharides-producing microorganism, asdescribed in Korean Laid-open publication No. 1989-1127, was used. Themodified composition is shown in Table 3 below. The microorganism wascultured in a 250 mL flask and the added amount of the medium was 50 mL.The culturing was performed in a agitating incubator with an agitationspeed of 150 rpm at 28° C. for 2 days.

TABLE 3 Ingredients Composition (g/L) Sucrose 200 NaNo₃ 2 K₂HPO₄ 5 Yeastextract 20 MgSO₄ 7H₂O 1 KCl 1 Note: pH corrected to 6.0 with 5N HCl

Next, the sucrose hydrolysis titer of fructosyl transferase which wasprepared from the cultured Penicillium citrinum KCTC 10225BP wasmeasured. The sucrose hydrolysis titer was measured according to themethod described by Shinohara Satoshi (Japanese Unexamined PatentApplication No. 10-165192). The enzyme titer was defined as an amount(μmol) of glucose produced by hydrolysis of sucrose, a sugar substrate,per unit time (minute). The fructosyl transferase prepared from themicroorganism Penicillium citrinum KCTC 10225BP according to the presentinvention was found to have a sucrose hydrolysis titer of 1.5 unit/1 gof sucrose on an average. Such a result demonstrates that fructosyltransferase derived from Penicillium citrinum KCTC 10225BP according tothe present invention has a sucrose hydrolysis titer superior to anyother known enzymes, considering the experimental results of U.S. Pat.No. 5,334,516, in which when hydrolyzing sucrose using an enzyme derivedfrom Aspergillus, at least 5 units of the enzyme was needed to degrade 1g of sucrose, and D. Grizard et al., in which 7 units of the enzymederived from Aspergillus awamori was needed to degrade 1 g of sucrose.

Also, upon examination of the sucrose hydrolysis titer according to thebiomass of the microorganisms, it was noted that as the biomass ofPenicillium citrinum KCTC 10225BP increases, degradation of sucrose isincreased.

EXAMPLE 3 Main Cultivation

The main cultivation (mass-production) was performed in a 5 L fermentor(Hanil R&D Co., Ltd. Korea) using the microorganism seed cultured inExample 2. The microorganism was inoculated in an amount of 5% (v/v) ofthe medium in the fermentor. The reaction conditions were a modificationof method described by Yu Moo-Young (Korean laid-open Publication No.1989-01127). FIG. 2 a shows the change in biomass of microorganismsaccording to the cultivation period upon varying the agitation speed ofthe agitator in the fermentor from 300 rpm to 500 rpm. As can be seen inFIG. 2 a, as the agitation speed was raised, the amount of themicroorganisms increased. FIG. 2 b shows the change in the amount ofintracellular and extracellular enzymes according the agitation speed.At the agitation speed of 500 rpm, the amount of intracellular enzymesshowed a tendency to decrease after 50 hours. It was believed that thiswas caused by aging of the microorganisms and deterioration of theenvironment in the fermentor. Similarly, at the agitation speed of 400rpm, the amount of intracellular enzyme was reduced after 50 hours.However, no increase of the amount of extracellular enzyme was observed.The increase of the amount of extracellular enzyme due to secretion bythe microorganism was expected to be about 20 units, considering thevolume of the fermentor, which was too tiny to be detected. FIG. 2 cshows the change in biomass of the microorganisms and the amount ofintracellular enzymes according to the fermentation time of the maincultivation using a fermentor at the agitation speed of 600 rpm. Overthe culturing time, the biomass of the microorganisms and the amount ofthe enzyme increased. When increasing the agitation speed of thefermentor, the biomass of the microorganisms showed a tendency toincrease. With respect to the morphology, micro-colloids were observedat an agitation speed of 400 rpm or more, and large pellets having adiameter of 2 were observed at an agitation speed of 200 rpm or less.

In terms of enzyme production, the amount of the enzyme present in themicroorganisms was 1400 units per 1 g of microorganism, and the amountof the enzyme existing outside of the microorganisms was 150 units per 1mL of the culturing medium. Therefore, it was shown that themicroorganism according to the present invention produced 2.8 times moreenzyme than the Penicillium citrinum FERM P-15944 of Shinohara et al.(Japanese Unexamined Patent Application No 10-165192), which containsthe enzyme in an amount of 500 unit/g.

EXAMPLE 4 Production of Fructooligosaccharides Using a Batch Type Method

Fructooligosaccharides were produced by reacting the microorganismsprepared in Example 2 with a sucrose solution. The totalfructooligosaccharides contents (solid %) was calculated by divisionwith the sum of the produced conventional fructooligosaccharides,neofructooligosaccharides, fructose, glucose and remained sucrose.

The change of the total fructooligosaccharides contents (solid %) weremeasured according to the agitation speed, culturing time, sucroseconcentration, pH and temperature change. In FIG. 3 a, the change in theamount of produced total fructooligosaccharides according to theconcentration of sucrose is shown. The highest production yield of theoligosaccharides was observed at a sucrose concentration of Brix 60 to70. When the concentration of sucrose was higher than Brix 70, sucrosewas crystallized as white precipitates, which interfered with thereaction with fructooligosaccharides. Therefore, the optimalconcentration of sucrose for industrial production is up to Brix 70.Meanwhile, with a low concentration of sucrose, the reaction can becontaminated by other microorganisms. Accordingly, it was found that theoptimal concentration of sucrose was Brix 65 to 70. In terms of thereaction time, in 24 hours, the solid ratio reached the maximum of 65%,which suggested that the present invention can be applied to practicalindustrial production without problems.

FIG. 3 b shows the change of the fructooligosaccharides contents (solid%) according to the present invention at pH ranging from 3 to 7. It wasnoted that at pH 5 to 7, the production yield of fructooligosaccharidesis high.

In the subsequent experiments, the sucrose solution was subjected toreaction without adjusting the pH of the reaction by addition of aseparate buffer solution. Therefore, the method for producingneofructooligosaccharides can be further simplified without anadditional treatment, in terms of industrial production.

FIG. 3 c shows the change in the total fructooligosaccharides contents(solid %) according to the reaction temperature. The totalfructooligosaccharides contents showed a tendency to constantly increaseup to 40° C. The highest reactivity was observed at 45° C. in terms ofthe reaction rate. The reaction was shown to be completed in 24 hours.It is expected that the problems associated with contamination by othermicroorganisms could be solved since the cultivation was performed at ahigh temperature. However, the total amount of the producedfructooligosaccharides was not significantly higher, although the enzymetiter at 45° C. was greater than those disclosed in other documents.This can be explained by a theory which has been proposed, stating thatproduction of oligosaccharides is inhibited by accumulation of glucoseproduced by hydrolysis of sucrose in the reactor.

As a solution for this, methods for consuming glucose have beensuggested, which include for example, a method for consuming glucose byaddition of glucose oxidase or yeast (See, Yoon, Jong-won, et al., TheBulletin of the Korean Society for Biotechnology and Bioengineering, 9,40-47, 1994). However, in the present invention, such method was notemployed, since impurities could be produced.

FIG. 3 d shows the change of the total fructooligosaccharides contents(solid %) according to the agitation speed. It was noted that thereactivity decreased as the agitation speed increased. It is believedthat the reaction area between the microorganisms and the sucrosesolution was decreased by bubbles generated when the agitator rotated ata high speed. Therefore, it was noted that the optimal agitation speedis up to 200 rpm. In the present invention, 100 rpm was used forproduction of fructooligosaccharides. As a consequence of performing theabove-described experiments for production of fructooligosaccharidescontaining neofructooligosaccharides, the optimal conditions forproducing fructooligosaccharides are defined as a sucrose concentrationof Brix 60 to 65, a reaction temperature of 45° C., a reaction time of24 hours and an agitation speed of 100 rpm.

EXAMPLE 5 Continuous Production of Fructooligosaccharides ViaImmobilization of Microorganism

Penicillium citrinum KCTC 10225BP seed cultured in Example 2 wasinoculated in an amount of 5% (v/v) of medium and cultivated in a 5 Lfermentor (Hanil R&D Co., Ltd. Korea). After completion of thecultivation, the microorganisms were collected and immobilized in sodiumalginate (Junsei Chemical, Japan) to produce fructooligosaccharides. Themicroorganisms at a certain concentration were mixed with the alginateat a certain concentration. The resulting mixture was flowed at apredetermined rate using a peristaltic pump (Gilson, France) and droppedthrough a needle having a diameter of 1 mm at a height of 20 cm to 1%aqueous calcium chloride (CaCl₂) solution, being stirred using a magnetstirrer. The 1% aqueous calcium chloride (CaCl₂) solution was used toform beads via ion exchange reaction between calcium ions (Ca²⁺) in thesolution and sodium ions (Na⁺) of the alginate. In order to determinethe optimal concentrations of microorganisms and the alginate to becombined, microorganisms at a concentration of 25 to 100 g/were mixedwith the alginate at a concentration of 1% to 2%. As a result, it wasfound that when microorganisms at a concentration up to 50 g/L weremixed with the alginate at a concentration up to 1.5%, perfectlyspherical beads were formed. Over the foregoing concentrations, beads ofnonuniform shapes were formed due to increase of viscosity. Therefore,in this example, microorganisms at a concentration of 50 g/L were mixedwith the alginate at a concentration of 1.5% to produce beads. Theresulting beads were stored at 4° C. for 10 hours, washed with distilledwater and dipped in a sucrose solution of Brix 60 at 4° C. for 10 hoursfor aging. Then, the beads were packed in a 1 L glass column and asucrose solution of Brix 60 were flowed through the column. Here, thereaction temperature was 45° C. and the sucrose solution was injected ata rate of 200 mL/hour by a peristaltic pump. FIG. 4 shows the change inthe amount of fructooligosaccharides and neofructooligosaccharidesproduced from the immobilized continuous method for 40 days. It wasnoted that solid contents of the total produced fructooligosaccharides(%) were constant within a range of 55% to 60% and both shape andhardness of beads were not different from the initial stage.

As a consequence of performing the above-described experiments forproduction of fructooligosaccharides containingneofructooligosaccharides, the optimal conditions for producingfructooligosaccharides by the immobilized continuous method were foundas a sucrose concentration of Brix 60, a reaction temperature of 45° C.,and a flow rate of the sucrose solution of 150 to 200 mL/hour.

EXAMPLE 6 Analysis of Fructooligosaccharides

The fructooligosaccharides obtained from Example 4 and Example 5 wereanalyzed using a high-performance liquid chromatography (HPLC) system(Shimadzu, Japan). A ODS column (5 μm, 150 mm×4 mm) produced by DaisoCo., Ltd. (Osaka, Japan) and a refractive index detector were used.Since there was detected a substance different from the conventionalfructooligosaccharides, the products were separated using a genuinepreparative high-performance liquid chromatography (Prep-HPLC) systemproduced by Waters Corp. (Massachusetts, USA). The resulting fractionswere subjected to a NMR analysis using an ARX 400 MHz NMR spectrometer(Bruker, Germany) to determine their structure. Results are shown inTable 4 below.

TABLE 4 Kestose Neo Kestose Nystose Neo Nystose Com- po- Compo- Compo-Compo- nent δC nent δC nent δC nent δC ¹F₁ 1 63.7 ⁶F₁ 1 63.00 ¹F₁ 163.82 ¹F₁ 1 62.99 2 106.7 2 106.41 2 106.0 2 106.36 3 79.4 3 79.58 379.6 3 79.58 4 76.6 4 77.13 4 76.6 4 77.10 5 84.0 5 83.86 5 84.0 5 83.826 65.0 6 65.08 6 64.98 6 65.03 ¹F₂ 1 63.2 ¹F₁ 1 64.25 ¹F₂ 1 63.63 ¹F₂ 163.77 2 106.5 2 106.43 2 105.8 2 105.97 3 79.4 3 79.04 3 80.3 3 79.19 477.2 4 76.70 4 77.2 4 76.52 5 83.9 5 84.06 5 83.8 5 83.91 6 65.1 6 65.126 65.04 6 64.90 G 1 95.3 G 1 94.72 ¹F₃ 1 63.16 ¹F₃ 1 63.17 2 73.9 273.73 2 106.4 2 106.42 3 75.4 3 75.17 3 79.5 3 79.36 4 72.0 4 71.94 477.1 4 77.20 5 75.20 5 74.26 5 83.8 5 83.82 6 62.9 6 63.08 6 65.03 665.03 G 1 95.3 G 1 94.97 2 73.9 2 73.75 3 75.4 3 75.26 4 72.0 4 71.90 575.2 5 74.27 6 62.89 6 63.03

As shown in the above table, it was confirmed that thefructooligosaccharides produced using Penicilliun citrinum KCTC 10225BPmicroorganism according to the present invention contained conventionalfructooligosaccharides (formula I) and also neofructooligosaccharides(formula II) having a structure different from that of the conventionalfructooligosaccharides. Therefore, it was proven that the Penicilliumcitrinum KCTC 10225BP microorganism used in the present invention canproduce conventional fructooligosaccharides simultaneously withneofructooligosaccharides having a different structure.

INDUSTRIAL APPLICABILITY

As described above, the present inventors identified a novelmicroorganism, Penicillium citrinum KCTC 10225BP, which can hydrolyzesucrose into the conventional fructooligosaccharides andneofructooligosaccharides. Fructosyl transferase prepared from thePenicillium citrinum KCTC 10225BP has a sucrose hydrolysis titer muchhigher than the conventional microorganism-derived enzymes Therefore, byusing Penicillium citrinum KCTC 10225BP, it is possible to producefructooligosaccharides along with neofructooligosaccharides in a highyield at a low cost.

1. An isolated and purified culture of Penicillium citrinum KCTC 10225BPhaving a fructosyl transferase that hydrolyzes sucrose intofructooligosaccharides of the following formula I:

in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, and neofructooligosaccharides of the followingformula II:

in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, using said fructosyl transferase.
 2. A method forproducing fructooligosaccharides and neofructooligosaccharidescomprising contacting sucrose with a purified and isolated Penicilliumcitrinum KCTC 10225BP having a fructosyl transferase that hydrolyzessucrose into fructooligosaccharides of the following formula I:

in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, and neofructooligosaccharides of the followingformula II:

in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, using said fructosyl transferase.
 3. A method forproducing fructooligosaccharides and neofructooligosaccharidescomprising: seed culturing in a first medium at 26° C. to 28° C. for 2days while agitating at a speed of 100 to 200 rpm a Penicillium citrinumKCTC 10225BP having a fructosyl transferase that hydrolyzes sucrose intofructooligosaccharides of the following formula I:

 in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, and neofructooligosaccharides of the followingformula II:

 in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, using said fructosyl transferase; mass-producingsaid microorganism in a fermentation medium at 26° C. to 28° C. for 72hours while agitating at a speed of 200 to 600 rpm and injecting air ata rate of 0.5 to 1 v/vm; and collecting the produced microorganisms bycentrifugation, washing the collected microorganisms twice with 0.85%physiological saline, and culturing in a sucrose solution having a Brixconcentration of 60 to 77 at a temperature of 35° C. to 50° C. and a pHof 5 to 7 for 20 to 50 hours at a speed of 100 to 300 rpm.
 4. A methodfor producing fructooligosaccharides and neofructooligosaccharidescomprising: seed culturing a Penicillium citrinum KCTC 10225BP having afructosyl transferase that hydrolyzes sucrose intofructooligosaccharides of the following formula I:

 in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, and neofructooligosaccharides of the followingformula II:

 in which n is an integer of 1 to 5, G represents glucose and Frepresents fructose, using said fructosyl transferase; mass-producingthe seed cultured microorganism; mixing the mass-produced microorganismswith a carrier to form beads; packing said beads in a column; andpassing a sucrose solution through said column.
 5. The method of claim4, wherein the seed culturing step is performed at 26° C. to 28° C. for2 days while agitating at a speed of 100 to 200 rpm.
 6. The method ofclaim 4, wherein the mass-producing step is performed at 26° C. to 28°C. for 42 to 72 hours while agitating at a speed of 200 to 600 rpm andinjecting air at a rate of 0.5 to 1 v/vm.
 7. The method of claim 4,wherein the carrier is one selected from a group consisting of alginategel, photo cross-linked resin, acrylamide gel, chitosan and gelatin. 8.The method of claim 4, wherein the carrier is used at a concentration of1 to 2%.
 9. The method of claim 4, wherein the beads are produced bydropping the mixture of the microorganisms and the carrier through aneedle having a diameter of 1 mm to 1 to 2% aqueous calcium chloride(CaCl₂) solution.
 10. The method of claim 4, wherein the sucrosesolution has a concentration of Brix 60 to
 70. 11. The method of claim4, wherein the sucrose solution is passed through the column at 35° C.to 55° C. and a rate of 100 to 300 mL/hour.